ESR SPIN-TRAPPING OF A PROTEIN-DERIVED TYROSYL RADICAL FROM THE REACTION OF CYTOCHROME-C WITH HYDROGEN-PEROXIDE

The reaction of horse heart cytochrome c with hydrogen peroxide was in vestigated using the ESR spin-trapping technique and the nitroso spin traps 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) and 2-methyl-2 -nitrosopropane (MNP). The ESR spectra obtained using both spin traps were typical of an immobilized nitroxide and indicated that the adduct was a macromolecule, The intensity of the ESR spectrum corresponding to the DBNBS/(.)cytochrome c radical adduct was greatly enhanced by pe rforming the reaction under anaerobic conditions, which suggested that the spin trap was competing with O-2 for reaction with the radical si te(s), Nonspecific proteolysis of either the DBNBS or the MNP adducts revealed isotropic three-line spectra, In addition, a high resolution ESR spectrum for the protease-treated MNP cytochrome c-derived protein radical adduct was obtained. The superhyperfine couplings detected in this spectra were identical to those detected from an authentic MNP/t yrosyl adduct, Carbon-13 labeling of the aromatic ring positions of ty rosine yielded additional hyperfine coupling, demonstrating that the r adical site was definitely located on the ring of tyrosine, Mass spect rometry detected as many as four DBNBS/(.)cytochrome c-derived adducts from the reaction of cytochrome with H2O2, Thus, it would appear four radical sites are formed during the reaction, at least one of which i s tyrosine.